Beware self-reacting fragments

Long-time readers will know that I have a peculiar fascination for artifacts of all kinds, particularly when they provide learning opportunities. A lovely example by Gerhard Klebe (Philipps-Universität Marburg) and collaborators has just appeared in Angew. Chem. Int. Ed.

The researchers have long been using the aspartic protease endothiapepsin (EP) as a model protein: we previously discussed how they compared half a dozen fragment-finding methods against EP, more recently arguing that crystallography is the best of the bunch. The new paper focuses on Compound 1. This molecule was a hit in five out of six fragment screens, each employing a different method, and was among the top ten hits in four of the screens. It produced the highest thermal shift (+3.4 °C), strongly inhibited the enzyme in two different biochemical assays, and even showed a dissociation constant of 115 µM by isothermal titration calorimetry (ITC).

Crystallography, though, told a different story. The researchers obtained high resolution structures (initially 1.25 Å, and ultimately 1.03 Å!) These revealed that the bound ligand was actually compound 2, which is composed of three molecules of compound 1. A variety of experiments, including anomalous scattering, high resolution mass spectrometry (HR-MS), and MS/MS fragmentation supports this assignment.

So what’s going on? The team notes that, although compound 1 does not aggregate and is not a PAINS compound, high-level quantum mechanical modeling suggested that the chlorine is susceptible to nucleophilic displacement, which probably wouldn’t surprise many medicinal chemists. Rearrangement of the resulting dimeric molecule produces compound 4, which could then react with yet another molecule of compound 1 through a radical mechanism to produce compound 2.

Allowing compound 1 to sit in buffer or methanol provided support for this mechanism and allowed the isolation of compound 4 and other degradation products, though compound 2 itself could not be detected. The researchers suggest it is particularly reactive and only stable when surrounded by the protein.

I applaud the investigators for pursuing this fascinating bit of science. This is academic research in the best sense of the phrase.

This is also the kind of investigation that would fall outside the scope of most industrial researchers, where the mandate is to discover promising drug leads as quickly as possible. More somberly, this story could have ended in embarrassment or worse had the researchers been less rigorous. The difficulty (and unlikelihood) of such lengthy investigations is why triaging shortcuts such as PAINS filters have been introduced, and why scientists using these tools must still be cautious: even molecules that aren’t PAINS can act through pathological mechanisms.

This is also why I believe that arguments that PAINS filters are inadequately defined and should thus be discarded are misguided. Sure, some PAINS molecules are drugs, and any rubric can be improved. But the nice thing about fragment screens is that they often produce a plethora of hits to pursue. A flawed triaging scheme will jettison some pearls among the pebbles, but without triage, far more resources will be lost chasing will-o'-the-wisps.